Rapid methods for identifying modifiers of cellular apoptosis activity

ABSTRACT

The invention provides a single-well, microscale method of determining the specific apoptotic activity of a cell. The method consists of contacting a cell population of about 1×10 5  cells for a time period of between about 30 minutes and 4 hours with a sufficient volume of medium containing an apoptotic specific diagnostic reagent and a diagnostic accessory reagent so as to cover the cell population, and determining the activity of the apoptotic specific diagnostic reagent. The invention also provides a method of identifying a compound which induces apoptosis. The invention further provides a rapid method of identifying a compound which inhibits apoptosis.

BACKGROUND OF THE INVENTION

[0001] This invention relates to the biological process of programmedcell death and, more specifically to rapid methods of measuringapoptotic activity and of screening for compounds which modulateapoptosis.

[0002] Cells can die by at least two fundamentally different biologicalprocesses. One process, termed necrosis, refers to cell or tissue deathwhich usually occurs as a result of massive physical or chemical insult.Necrosis is characterized, in part, by cell swelling organelledisintegration and the leakage of cell cytoplasm into the extracellularspace and is generally considered to be a passive process.

[0003] The alternative process of cell death is known as apoptosis orprogrammed cell death. This mechanism of cell death in mammalian cellsis characterized by a set of morphological and biochemical changes thatreflect an active cell suicide. Apoptotic changes include cellshrinkage, nuclear chromatin condensation and margination, and DNAfragmentation. Biochemical events include the externalization ofphosphatidyl serine and the activation of aspartate-specific cysteineproteases.

[0004] In regard to the latter biochemical event, proteases within thecysteine aspartic acid protease family, also known as the ICE/CED-3family, are critical for effecting the process of apoptosis. Theseenzymes are cysteine proteases and exhibit substrate specificity forcleavage after an aspartic acid residue. Due to these characteristics,these enzymes are now referred to by the above term “cysteine asparticacid proteases” or “caspases”. During the apoptotic process, caspaseactivity is generated in cells and known inhibitors of the caspasefamily of proteases inhibit apoptosis.

[0005] Apoptosis is important clinically for several reasons. In thefield of oncology, many of the clinically useful drugs kill tumor cellsby inducing apoptosis. For example, cancer chemotherapeutic agents suchas cisplatin, etoposide and taxol all induce apoptosis in target cells.In addition, a variety of pathological disease states can result fromthe failure of cells to undergo proper regulated apoptosis. For example,the failure to undergo apoptosis can lead to the pathologicalaccumulation of self-reactive lymphocytes such as that occurring in manyautoimmune diseases, and can also lead to the accumulation of virallyinfected cells and to the accumulation of hyperproliferative cells suchas neoplastic or tumor cells. The development of efficacious compoundswhich are capable of specifically inducing apoptosis would therefore beof therapeutic value in the treatment of these pathological diseasesstates.

[0006] In contrast, the inhibition of apoptosis is also of clinicalimportance. For example, cells are thought to die by apoptosis in thebrain and heart following stroke and myocardial infarction,respectively. Moreover, the inappropriate activation of apoptosis canalso contribute to a variety of other pathological disease statesincluding, for example, acquired immunodeficiency syndrome (AIDS),neurodegenerative diseases and ischemic injuries other than those listedabove. As apoptotic inducers are of benefit in the previously mentioneddisease states, specific inhibitors of apoptosis would similarly be oftherapeutic value in the treatment of these latter pathological diseasestates.

[0007] Drug discovery benefits from the use of efficient high throughputmethods which can rapidly identify specific molecules that interact withthe target of interest. The identification of compounds whichspecifically modulate the apoptotic pathway so far has been hindered bythe lack of such methods. Available methods are either limited by thelack of specificity and/or efficiency. For example, most anti-cancerdrugs are screened for their ability to kill cells and therefore willidentify compounds that induce both necrosis or apoptosis. Moreover,many of these methods are often cumbersome in that they requireassessment of cell viability and take days to perform.

[0008] Attempts have been made to create methods that are specific forapoptosis. For example, the amount of DNA degradation induced in a cellpopulation has been used for a measurement of apoptosis. However, DNAdegradation is a relatively late step in the apoptotic process.Moreover, DNA degradation is not truly specific for apoptosis since DNAeventually becomes degraded in necrotic cells too.

[0009] Other methods currently employed use metabolic determinations asan attempt to measure apoptosis in a relatively shorter time frame. Forexample, cells undergoing apoptosis show impaired mitochondrial functionwhich can be measured using dyes such as alamar blue or by colourimetricassays such as reduction of MTT (3-(4.5-dimethyl)thiazol-2-yl-2,5-diphenyl tetrazolium bromide) to formazan. However,impaired mitochondrial function is not specific for apoptosis as it isalso a characteristic exhibited by necrotic cells.

[0010] There has been one method described where apoptotic measurementsappear specific and are conducted in relatively short time periods. Forexample, the successful measurement of caspase activity by measuring thefluorescent cleavage product of the CPP32 substrate analog DEVD-AMC hasbeen reported (Armstrong et al. J. Biol. Chem, 271:16850-16855 (1996)).However, this method required separate preparations of the cell lysateand reaction mixture as well as additional manipulations, includingsample washing in the assay procedure. In addition to the extra timerequired to perform these additional manipulations, this method couldnot be performed in a single step due to the requirement for separatepreparation of lysate and reaction mixture. Therefore, regarding highthroughput assays for apoptosis, what benefit might have been gained inspecificity was lost due the inefficiencies incurred in order to measurethe caspase activity.

[0011] Another method specific for apoptosis has been reported whereseparate preparations of cell lysate and reaction mixture has not beenrequired (Los et al. Nature 375:81-83 (1995)). This method similarlydetermined the caspase activity following induction of apoptosis bymeasuring the cleavage product of an ICE substrate analogue. Separatepreparation of samples and reaction was avoided due to the use of adetergent which does not completely lyse the cells. However, theincomplete solubilization of cellular components can result in decreasedsensitivity of the method. Moreover, the detection of substrate cleavagewas performed by a separate procedure and, as with the method ofArmstrong et al. above, similarly required additional manipulations andsteps which lengthened the time period for the procedure.

[0012] Thus, there exists a need for rapid and efficient methods toidentify compounds which can specifically modulate the apoptotic pathwayfor the therapeutic treatment of human diseases. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0013] The invention provides a single-well, microscale method ofdetermining the specific apoptotic activity of a cell. The methodconsists of contacting a cell population of about 1×10 cells for a timeperiod of between about 30 minutes and 4 hours with a sufficient volumeof medium containing an apoptotis-specific diagnostic reagent and adiagnostic accessory reagent so as to cover the cell population, anddetermining the activity of the apoptotis-specific diagnostic reagent.The invention also provides a method of identifying a compound whichinduces apoptosis. The method consists of (a) providing a cellover-expressing a cell survival polypeptide at a level which issufficient to prevent the induction of apoptosis; (b) treating the cellover-expressing the cell survival polypeptide with a direct stimulus ofthe cell death pathway; (c) adding a compound to be tested for apoptoticinducing activity, and (d) determining cellular apoptotic activity, thepresence of which is indicative of the compound being an apoptoticinducer. The invention further provides a rapid method of identifying acompound which inhibits apoptosis. The method consists of (a) separatelycontacting a plurality of cell populations with a different compound tobe tested for apoptotic inhibiting activity; (b) incubating the cellswith a direct stimulus of the cell death pathway for a period of betweenabout 2 minutes to 3 hours, and (c) measuring the specific apoptoticactivity of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows a comparison of the single-well method of measuringspecific apoptotic activity with a method requiring additional washingprocedures. FIG. 1A shows the caspase activity induced inneo-transfected Jurkat cells in the presence (closed symbols) or absence(open symbols) of anti-Fas antibody. FIG. 1B shows the inhibition ofinduced caspase activity in Bcl-2 transfected cells in the presence orabsence of anti-Fas anti-body. (: wash+anti-Fas; ◯:wash−anti-Fas;▪:single-well+anti-Fas; □:single-well-anti-Fas)

[0015]FIG. 2 shows the induced caspase activity measured at differenttime points following treatment with a range of anti-Fas antibodyconcentrations. FIG. 2A shows the anti-Fas antibody titration andcaspase time course in neo-transfected Jurkat cells. FIG. 2B shows aparallel titration and time course in Bcl-2 transfected Jurkat cells.

[0016]FIG. 3 shows the screening of a chemical compound library usingBcl-2 transfected cells primed with anti-Fas antibody and theidentification of positive apoptotic inducers.

DETAILED DESCRIPTION OF THE INVENTION

[0017] This invention is directed to a novel method for the rapid andefficient measurement of apoptosis. The method is advantageous in thatit distinguishes apoptosis from other forms of cell death and can beperformed in a single step and on a small scale analysis. Moreover, themethod is rapid in that it can be performed in a time period of betweenabout 30 minutes and 4 hours. These advantages allow for the highthroughput screening of a large number of samples with a specificity andtime period not achieved before with previous methods known in the art.The method distinguishes apoptosis from other forms of cell death inthat it measures the activity of cysteine aspartic acid proteases orother specific indicators of apoptosis. Therefore, the method can beadvantageously used for the specific identification of inducers ofapoptosis such as compounds that inhibit the function of Bcl-2.

[0018] In one embodiment, the invention is directed to a method ofmeasuring apoptosis where a single buffer is added which contains alysis agent and a detectable substrate for one or more aspartatespecific cysteine proteases. A specific example of a detectablesubstrate is the peptide analogue DEVD-AMC which fluoresces afterprotease cleavage. Simultaneous lysis with substrate allows for therapid measurement of aspartate specific cysteine protease activitywithout the necessity for washes or other transfer of reagents.

[0019] In another embodiment, the invention is directed to a method foridentifying compounds which induce apoptosis. The method employs themeasurement of induced cysteine aspartic acid protease activity in acell line that expresses the Fas antigen and has been engineered tooverexpress the cell survival polypeptide Bcl-2 and which has beentreated with anti-Fas antibody. In a normal unengineered cell line,treatment with the anti-Fas antibody causes rapid apoptosis. However, inthe engineered cell line apoptotic progression is blocked due to thecontinued presence of Bcl-2. Compounds that induce apoptosis areidentified by incubating them with the engineered cell line that hasals6 been incubated with anti-Fas antibody and then measuring apoptosisusing the method described above. Due to the overexpression of Bcl-2,this method for identifying inducers of apoptosis will identifycompounds that either inhibit the function of Bcl-2, or that stimulatethe cell death pathway downstream of the Bcl-2 blockade. Moreover, sincethe engineered cells are treated with the pro-apoptotic anti-Fasantibody, these cells are primed for programmed cell death so that thereis little or no lag time once the cells are treated with a positiveapoptotic inducer. This priming of the cells provides additionalsensitivity and speed for identifying apoptotic inducers. The primingfor cell death can be provided by other pro-apoptotic stimuli includingbut not limited to, staurosporine, TNF and TNF plus cycloheximide.

[0020] In another embodiment, the invention is directed to a method ofidentifying inhibitors of apoptosis. This method also utilizes thesingle-step method described above, however, the indicator cell linedoes not require the overexpression of a cell survival polypeptide.Instead, the cells are first incubated with a compound to be tested forinhibitory activity and then treated with an direct stimulus ofapoptosis such as anti-Fas antibody. In the absence inhibitorycompounds, cells subsequently treated with anti-Fas antibody undergoapoptosis and exhibit a rapid induction of aspartate specific cysteineprotease activity. In contrast, positive compounds inhibit the emergenceof protease activity compared to control samples.

[0021] As used herein, the term “apoptosis” is intended to mean thephysiological process known as programmed cell death. This process is amorphologically and biochemically distinct form of cell death thatregulates cell turnover under normal physiological conditions. Themorphological features include an orchestrated sequence of changes whichinclude cell shrinkage, chromatin condensation, nuclear segmentation andeventual cellular disintegration into discrete membrane-bound apoptoticbodies. The biochemical features include, for example, internucleosomalcleavage of cellular DNA and the activation of ICE/Ced-3 family ofproteases. The term “apoptosis” is used here synonymously with thephrase “programmed cell death.”

[0022] These terms are intended to be consistent with their use as theyare known and used by those skilled in the art.

[0023] As used herein, the term “single-well” when used in reference toan apoptotic assay method is intended to mean that all actions,processes, or measures taken to achieve the determination of specificapoptotic activity are performed without the need for intermediaryprocessing steps that require the cells to be moved from one well orvessel to another. Such intermediary processing steps can include, forexample, cell harvesting; washing steps; purification steps; separationsteps; exchange of additional buffers or reagents; media, reagent orlysate transfer; or, sample transfer or processing for analysis. Theterm includes, however, the addition or mixing of other buffers orreagents. Thus, the term “single-well” is intended to mean that themethod can be performed by adding to a cell sample all reagentsnecessary to determine the specific apoptotic activity following anappropriate time of incubation. The addition of such reagents can beperformed in a single step or they can be added sequentially.Measurement of specific apoptic activity therefore occurs withoutfurther processing or transfer of the sample. The term single-well isalso intended to include scalable formats and automated procedures.

[0024] As used herein, the term “microscale” is intended to mean thatthe method can be performed on a scale measured in microliter (μl) orsub-microliter volumes and whereby many sample measurements can be madein parallel in, for example, multi-well plates. Such a scale is incontrast to milliters (ml) and to procedures not amenable to amulti-well format. A specific example of a multi-well format is a96-well ELISA plate. The microscale measurable volumes are between about1 and 200 μl, more preferably between about 30 and 125 μl, andpreferably about 100 μl. Thus, a microscale method can be entirelyperformed, for example, in a microwell plate format such as a 96 well orother multiwell sample format.

[0025] As used herein, the term “specific apoptotic activity” isintended to mean cellular activity specifically due to the activation ofthe programmed cell death pathway. Programmed cell death is effected andregulated by a variety of molecules including the Bcl-2 and Baxpolypeptides as well as the activation of members of the ICE/Ced3-familyof cysteine aspartic acid proteases (caspases). Thus, cellular activityspecifically due to the activation of the programmed cell death pathwayis intended to mean the activity of molecules, including the abovefamilies of molecules, which regulate or participate in the apoptosispathway and whose activity correlates with the appearance of apoptosisin these cells.

[0026] As used herein, the term “direct stimulus” when used in referenceto the cell death pathway is intended to mean an agent that increasesthe specific apoptotic activity of a cell. Specific examples of directstimuli include, for example, Fas ligand, anti-fas antibody,staurosporine, ultraviolet (UV) and gamma irradiation. Other directstimuli exist and are known by those skilled in the art. Thus, a directstimulus of apoptosis is an agent which increases the molecular activityof the above-described families of molecules which enhance orparticipate in apoptosis.

[0027] As used herein, the term “apoptotic specific diagnostic reagent”or “diagnostic reagent” is intended to mean a reagent which specificallymeasures or can be made to specifically measure the specific apoptoticactivity of a cell. Such reagents include, for example, the measurementof caspase activity. Moreover, such measurements can be either direct orindirect. Apoptotic specific diagnostic reagents can include substrateanalogues for caspase family members which fluoresce upon cleavage.Specific examples of such substrate analogues include, for example,ZEVD-AMC, YVAD-AMC and DEVD-AMC(carbobenzoxy-Glu-Val-Asp-aminomethylcoumarin, Tyr-Val-Ala-Asp-AMC andAsp-Glu-Val-Asp-AMC, respectively).

[0028] Reagents other than those which measure the binding or activityof cell survival or cell death polypeptides are similarly includedwithin the definition of the term so long as such reagents canspecifically measure, or be made to specifically measure apoptoticevents. An example of such an apoptotic specific diagnostic reagent isthe phospholipid binding polypeptide Annexin V. This reagent bindsphosphatidylserine in a calcium dependent manner. Specificity ofphosphatidylserine as a measure for apoptotic cells is due to the factthat this lipid is primarily found on the inner surface of a cellmembrane but is translocated to the outer surface upon cell death. Itsuse in conjunction with a viability stain can provide specificity forapoptotic mediated cell death. Thus, the measurement ofphosphatidylserine on the outer membrane surface can be used as anapoptotic specific diagnostic reagent. Other specific indicators ofapoptosis exist and are known to those skilled in the art. Such otherspecific indicators are intended to be include within the definition ofthe term as defined and used herein.

[0029] As used herein, the term “diagnostic accessory reagent” or“accessory reagent” is intended to mean an agent that is required forthe apoptotic specific diagnostic reagent to function. The agent can bea single ion, an organic or inorganic molecule, a macromolecule or anycombination thereof. The requirement for function of the diagnosticreagent can be biochemical or physical. A specific example of abiochemical requirement is found with the case of Annexin V wherecalcium is necessary for phospholipid binding. A physical requirementfor an accessory reagent is in the case where caspase activity ismeasured. In order to measure caspase activity, either the cell has tobe lysed or the substrate analogue has to permeate the membrane. Theaccessory reagent would facilitate such a lysis or crossing of themembrane. Specific examples of such accessory reagents would be an agentwhich is capable of lysing the cell or disintegrating the cell membrane,or alternatively, an agent which punctures holes in the membrane toallow for molecules and macromolecules to cross the membrane. Suchagents are known to those skilled in the art and are intended to bewithin the scope of the definition as it is used herein.

[0030] The term “lysis reagent” as it is used herein is thereforeintended to mean a reagent which is capable of causing the dissolutionor destruction of cellular membrane integrity. Such reagents generallyinclude, for example, detergents such as SDS, NP-40 and triton, however,lysis reagents other than detergents are also included within thedefinition so long as such reagents do not interfere with the cellularapoptotic activity and detection using an apoptotic specific diagnosticreagent. Additionally, the meaning of the term is also intended toinclude detergents or other reagents which do not result in cellulardestruction, but instead, are capable of permeabilizing the membrane andallowing entry of the diagnostic reagent. A specific example, of such alysis reagent includes digitonin.

[0031] As used herein, the term “compound” when used in reference to anagent capable of inducing or inhibiting apoptosis is intended to mean amolecule able to modulate the specific apoptotic activity of a cell.Such a molecule can include small organic or inorganic molecules as wellas large macromolecules. Specific examples of small molecules includeetoposide and carbobenzoxy-Val-Ala-Asp-fluoromethylketone. Examples ofmacromolecules which are able to modulate the specific apoptoticactivity of a cell include peptides, polypeptides, proteins, nucleicacid, carbohydrate and lipid. Functional or structural analogues ormimics of such compounds which exhibit substantially the same activationor inhibition activity are also included within the meaning of the termas used herein. The type, size or shape of the molecule is not importantso long as the molecules can either induce or inhibit the specificapoptotic activity of a cell.

[0032] As used herein, the term “cell survival polypeptide” is intendedto mean a peptide, polypeptide or protein that is capable of inhibitingthe specific apoptotic activity of a cell. Cell survival polypeptidesincludes those polypeptides which directly regulate the programmed celldeath pathway such as Bcl-2, Bcl-xL, Mcl-1 and the ElB-19K protein ofadenovirus as well as those which indirectly regulate the programmedcell death pathway.

[0033] The meaning of the term “cell survival polypeptide” also includesfunctional fragments so long as they have the ability to inhibit thespecific apoptotic activity of a cell. The term is also intended toinclude polypeptides that include, for example, modified forms ofnaturally occurring amino acids such as D-steroisomers, non-naturallyoccurring amino acids, amino acid analogues and structural mimics solong as such polypeptides retain functional activity as defined above.

[0034] As used herein, the term “overexpressing” when used in referenceto the level of a cell survival polypeptide is intended to mean anincreased accumulation of the cell survival polypeptide in theoverexpressing cells compared to their levels in counterpart normalcells. Overexpression can be achieved by natural biological phenomenonas well as by specific modifications as is the case with geneticallyengineered cells. Overexpression also includes the achievement of anincrease in cell survival polypeptide by both endogenous or exogenousmechanisms. Overexpression by natural phenomenon can result by, forexample, a mutation which increases expression, processing, transport,translation or stability of the RNA as well as mutations which result inincreased stability or decreased degradation of the polypeptide. Suchexamples of increased expression levels are also examples of endogenousmechanisms of overexpression. A specific example of a natural biologicphenomenon which results in overexpression by exogenous mechanisms isthe adjacent integration of a retrovirus. Overexpression by specificmodification can be achieved by, for example, the use of recombinantmethods known in the art to construct and overexpress stably ortransiently a cell survival polypeptide in a compatible vector-hostsystem.

[0035] As used herein, the term “cell death polypeptide” is intended tomean a peptide, polypeptide, or protein that is capable of increasing orinducing the specific apoptotic activity of a cell. Cell deathpolypeptides includes those polypeptides which directly regulate theprogrammed cell death pathway such as Bax, Bad, Bcl-xS, Bak, and Bik aswell as those which indirectly regulate the programmed cell deathpathway. Cell death polypeptides also includes, for example,ICE/Ced3-family of caspases since such caspases can induce programmedcell death.

[0036] The meaning of the term “cell death polypeptide” also includesfunctional fragments so long as they have the ability to enhance orinduce the specific apoptotic activity of a cell. As with functionalfragments of the counterpart cell survival polypeptides, the term isalso intended to include polypeptides that include, for example,modified forms of naturally occurring amino acids such asD-steroisomers, non-naturally occurring amino acids, amino acidanalogues and structural mimics so long as such polypeptides retainfunctional activity as defined above.

[0037] As used herein, the term “aspartate specific cysteine protease”or “caspase” is intended to mean those family of proteases which arerelated genetically to the C. elegans ced-3 gene product and includes,for example, human ICE (interleukin-1-β converting enzyme), ICH-1_(L),CPP32, Mch2, Mch3, Mch4, Mch5, Mch6, ICH-2 and ICE_(rel)-III. Inaddition to the shared homology to Ced3, the known caspases also sharethe following characteristics: 1) they exhibit cysteine proteaseactivity with specificity for substrate cleavage at Asp-x bonds, 2)contain a conserved pentapeptide sequence (QACRG, QACGG or a relatedsequence) within the active site and 3) are synthesized as proenzymesthat require proteolytic cleavage at specific aspartate residues foractivation of protease activity. Although these proteases are definedherein as cell death polypeptides, several alternative structural formsof caspases exist, such as ICEδ, ICEε, ICH-1_(S) and Mch2β, whichfunction to inhibit apoptosis. These alternative forms essentially actas dominant negative mutations and therefore are considered to be cellsurvival polypeptides.

[0038] The invention provides a single-well, microscale method ofdetermining the specific apoptotic activity of a cell. The methodconsists of contacting a cell population of about 1×10⁵ cells for a timeperiod of between about 30 minutes and 4 hours with a sufficient volumeof medium containing an apoptotic specific diagnostic reagent and adiagnostic accessory reagent so as to cover the cell population, anddetermining the activity of said apoptotic specific diagnostic reagent.

[0039] The method of the invention provides significant advantages overmethods known in the art in that it is rapid, efficient and is specificfor apoptosis. The speed and efficiency are attributed at least to therequirement of all steps being carried out without removing cells from asingle microwell for the measurement of apoptotic activity. Specificitycan be attributed at least to the measurement of apoptotis-specificbiochemical events.

[0040] In regard to the speed and efficiency, separate procedures arenot required for cell lysis and assay conditions. Moreover, followingthe appropriate incubation time for specific apoptotic events to occur,the method is absent of further washing procedures, transfers toadditional containers and/or subsequent manipulations required forindependent measurement procedures. Thus, the method of the inventionessentially takes a sample of cells and adds a single solution whichcontains all reagents necessary for the specific detection of apoptosis,incubates the mixture and then measures the apoptotic activity withinthe mixture.

[0041] Apoptosis can be measured in a variety of cell types, sources andformats. Usually the cell sample will be, for example, a eukaryotic cellline or other culturable cell type and is plated into a tissue culturedish or multiwell plate treated or untreated to allow analysis of thecell sample. The cell sample can be, for example, an adherent cell typeor one which grows in suspension. Other types of cell samples such astissues are similarly amenable for use in the method of the invention.Those skilled in the art will know, or can determine, the appropriateconditions which are necessary to enable practicing of the method onvarious types of cell samples.

[0042] The cell number utilized in the methods of the invention can bevaried depending on the cell type and which diagnostic reagent will beused to determine specific apoptotic activity of the cell. For example,the Jurkat T-cell line expresses relatively high levels of caspaseactivity and fewer than 100,000 cells are required to obtain an accuratemeasurement. Cells which express lower levels of caspase activitycompared to the Jurkat line can also be used. However, a larger cellpopulation will necessarily be employed. Those skilled in the art willknow or can determine the relative levels of caspase activity in aparticular cell sample and will therefore know the quantity of cellsrequired for determining specific apoptotic activity.

[0043] Therefore, the invention provides the measurement of specificapoptotic activity in a cell population with as few as 10,000 and asmany as 1×10⁶ cells. Usually the cell population is greater than about50,000 to allow for relatively simple detection and preferably the cellpopulation is about 100,000. Of course, the cell population can befurther decreased compared to those sizes given above, and the methodcan be compensated for by allowing a slightly longer incubation timewith the apoptotic stimulus, detection reagent and/or both. Thus, thereare a variety of alternatives available to those skilled in the artwhich can be employed to practice the invention.

[0044] Reagents necessary for apoptosis include an apoptotic specificdiagnostic reagent and an accessory reagent. The diagnostic reagent willvary depending on the specific apoptotic event that will be measured.For example, caspase activity can be used as a marker of specificapoptotic activity. Activity can be determined using, for example,caspase substrate analogues which contain a detectable moiety. Althougha variety of different labels can be utilized for the detectable moiety,for use in the single-well method of the invention, the detectablemoiety should have a different property following cleavage of substratewhen compared to the starting substrate analogue. The change inproperties for the label allows for measurement of specific apoptoticactivity without the need for additional steps to be performed in theassay to remove, for example, unreacted or unbound substrates fromproducts. Thus, diagnostic reagents which contain detectable moietiesthat fluoresce or luminesce, for example, upon cleavage are amenable foruse in the single-well method of the invention. Specific examples ofsuch caspase substrate analogues containing detectable moieties thatfluoresce upon cleavage include the peptide analogues ZEVD-AMC, YVAD-AMCand DEVD-AMC. Alternatively, detectable moieties which fluoresce at adifferent wavelength following cleavage or even following binding to acaspase are also amenable for use in the methods described herein.

[0045] Apoptotic specific diagnostic reagents other than caspasesubstrates exist as well and can alternatively be used in the methods ofthe invention. A specific example of such other diagnostic reagentsincludes the phospholipid binding polypeptide Annexin V. As with theabove-described caspase substrate analogues, these other diagnosticreagents can similarly be used in the methods of the invention becausethey measure a specific apoptotic event, or can be made to measure aspecific apoptotic event, and also do not require additional proceduresor manipulations in order to obtain a positive indication of apoptoticactivity.

[0046] Annexin V, for example, has a high affinity forphosphatidylserine which is translocated to the outer membrane duringcell death. Binding of Annexin V is therefore a measure of apoptoticactivity. Phospholipid binding can be determined by, for example, FITClabeled Annexin V followed by removal of unbound label by filtration.The use of sample wells having bottoms comprising membrane filters canbe used in conjunction with Annexin V as a diagnostic agent sincefiltration and subsequent analysis can occur without the need for sampletransfer or additional processing steps. Diagnostic reagents other thanAnnexin V exist and are known to those skilled in the art. Such otherreagents can similarly be used in the methods of the invention given theteachings provided herein.

[0047] An accessory reagent is also required for use in the claimedmethods of the invention. The accessory reagent will vary depending onthe type of diagnostic reagent used. For example, when using a caspasesubstrate analogue as the diagnostic reagent, the accessory reagent willbe an agent that allows for the lysis, solubilization orpermeabilization of the cell membrane. Such procedures will eitherliberate the cytoplasmic contents of a cell to allow for measurement ofcaspase activity or allow transport of the substrate analogue across thelipid bilayer. Reagents other than those that destroy or permeabilizethe membrane can be employed as well so long as they allow thecolocalization of the diagnostic reagent with the particular molecule oractivity to be measured. For example, lipid vesicles or other deliveryparticles as well as receptor mediated events can similarly be used asthe accessory reagent for colocalizing the diagnostic reagent with thecytoplasmic contents of an apoptotic cell.

[0048] Selection of an accessory reagents will vary depending on theparticular application and/or apoptotic event that will be measured.Accessory reagents have been described above in reference to caspaseactivity for the measurement of cytoplasmic apoptotic events. However,alternative accessory reagents can be employed for the measurement ofapoptotic events on the cell surface or cell exterior. In this regard,it is not necessary to bring together the cytoplasmic contents and thediagnostic reagent if the apoptotic event is localized on the cellsurface or exterior. In this particular case, the accessory reagent canbe, for example, an agent which allows for better access of thediagnostic reagent to the cell surface or exterior.

[0049] In addition, accessory reagents can also be, for example,cofactors or other compounds which enable functioning of the diagnosticreagent. For example, the diagnostic reagent Annexin V is a calciumdependent phospholipid binding polypeptide. An accessory reagent forAnnexin V can therefore be the presence of calcium in the medium tofacilitate binding of the diagnostic reagent with phosphatidylserine onapoptotic cells. In some instances there may not be a requirement for areagent to facilitate measurement or colocalization of the diagnosticreagent. In these particular instances, the accessory reagent can be,for example, the buffer in which the diagnostic reagent works optimally.

[0050] Additionally, an accessory reagent can be more than one reagentpresent in the medium containing the diagnostic reagent. For example, anaccessory reagent can have two components such as a lysis reagent and acofactor or other agent which facilitates measurement of apoptoticactivity. Moreover, reagents which enhance the specificity of thediagnostic reagent can additionally be included as an accessory reagent.Dyes which measure cell viability are specific examples of suchaccessory reagents which enhance specificity. Those skilled in the artwill know or can determine what combinations of agents should be used asan accessory reagent. Thus, the choice of the accessory reagent willdepend on the diagnostic molecule and the apoptotic event to bemeasured. Those skilled in the art will similarly know which accessoryreagents are applicable with which diagnostic reagents given theteachings described herein.

[0051] Depending on the cell type, number and diagnostic reagent used,incubation time periods will vary according to the need and the specificapoptotic activity to be measured. For example, using Jurkat cells, andmeasuring for the induction of caspase activity as little as a 30 minuteincubation period with the diagnostic reagent is required for detectionof a signal. This period of incubation is measured after, for example,the induction of apoptosis. For the specific example, of caspaseactivity, enzyme production of product will increase over time and willsimilarly lead to an increase in signal. Therefore, longer incubationperiods with the diagnostic reagent will lead to stronger signals. Otherfactors can be adjusted to modify the incubation time needed todetermine the specific apoptotic activity of a cell. For example, cellnumber or reagent concentrations can be increased to decrease theincubation time necessary to obtain a reliable measurement. Suchmodifications are known to those skilled in the art or can be routinelydetermined by testing various cell numbers and reagent concentrationsover several time points to determine those modifications which provideoptimal performance for the particular need. Normally, not more than 6hours is required for detection of apoptotic activity, preferablybetween about 30 minutes and 2 hours and usually not more than about 1hour of incubation with a diagnostic reagent is necessary for measuringthe specific apoptotic activity of a cell.

[0052] As with cell type, cell number, reagent concentrations andincubation times, so can medium volumes be varied to obtain a desiredresult or to fit a particular need. For example, medium volumessufficient to cover the cell sample should be used so as to allowadequate measurement of apoptotic activity. Volumes can be adjustedaccording to the solubility properties of the diagnostic reagent and theaccessory reagent. Essentially, all that is necessary is for the mediumto contain the appropriate amounts of required reagents and to make thereagents available to the molecules to be measured. Preferably thevolumes should be confined to that of a microwell chamber such as a96-well ELISA plate. Such volumes are generally between about 1 and 200μl, more preferably between about 30 and 125 μl and preferably about 100μl. Those skilled in the art will know what volumes are useful for theparticular situation.

[0053] Thus, the method is amenable to a multiwell format assay wherelarge numbers of samples can be screened rapidly and efficiently. Inparticular, a 96 well format provides practical advantages since platesappropriate for manipulations and measuring devices are commerciallyavailable. Such procedures can be further automated to increase furtherthe speed and efficiency of the method. These features, combined withthe specificity of the method, allows for the high throughput screeningof compounds which either induce or inhibit apoptosis. For example, alibrary of test compounds can be administered to a plurality of cellsample populations and then assayed for their ability to induce orinhibit apoptosis. Each of the different test compounds is administeredfor a sufficient time so as to induce or inhibit apoptosis prior todetermining specific apoptosis activity. Such an incubation time isusually about 2 minutes to 4 hours. Identified compounds are valuablefor both therapeutic and diagnostic purposes since they can allow forthe treatment and detection of apoptotic mediated diseases. Suchcompounds are also valuable in research related to apoptotic mechanismssince they can help deduce further molecular events and provide furtherspecificity for the discovery and development of future compounds.

[0054] The single-well microscale method described previously fordetermining the specific apoptotic activity of a cell can also beemployed for the rapid screening and identification of inhibitors ofapoptosis. Therefore, the invention also provides a rapid method ofidentifying a compound which inhibits apoptosis. The method consists of(a) separately contacting a plurality of cell populations with adifferent compound to be tested for apoptotic inhibiting activity; (b)incubating said cells with a direct stimulus of the cell death pathwayfor a period of between about 2 minutes to 3 hours, and (c) measuringthe specific apoptotic activity of the cells.

[0055] The invention further provides a method of identifying a compoundwhich induces apoptosis. The method consists of (a) providing a celloverexpressing a cell survival polypeptide, the cell survivalpolypeptide being overexpressed at a level which is sufficient toprevent the induction of apoptosis; (b) treating the cell overexpressingthe cell survival polypeptide with a direct stimulus of the cell deathpathway; (c) adding a compound to be tested for apoptotic inducingactivity, and (d) determining cellular apoptotic activity, wherein thepresence of apoptotic activity is indicative of the compound being anapoptotic inducer.

[0056] The method described above for the identification of inducers ofapoptosis is particularly valuable providing an enhanced format formeasuring apoptotic activity in that a cell is treated so that it is“poised” for programmed cell death. In this way the cell has synthesizedand/or activated all necessary components that are required forprogrammed cell death. All that is necessary is a stimulus to push thecell past its holding point and into apoptosis. A positive test compoundis just that stimulus to cause the cell to progress into programmed celldeath.

[0057] The holding point which prevents the cell from proceeding intoprogrammed cell death is the overexpression of a cell survivalpolypeptide. Cell survival polypeptides are characterized in that theyexhibit the ability to prevent apoptosis when expressed or activated ina cell induced to undergo apoptosis. For example, in the absence of afunctioning cell survival polypeptide, a cell treated with an apoptoticinducer will initiate the programmed cell death pathway and eventuallydie by apoptosis. However, in the presence of a cell survivalpolypeptide, treatment with an apoptotic inducer can initiate theprogrammed cell death pathway but the cell will survive due toinhibition of one or more events along the pathway. Depending upon thepoint at which the cell survival polypeptide functions, the programmedcell death pathway can be inhibited early or relatively late within theexecution of the cascade of events leading to ultimate cell death. Cellsurvival polypeptides and their encoding nucleic acids are well known inthe art and include, for example, the Bcl-2 family of related proteinsBcl-2, Bcl-xL, Mcl-1, E1B-19K as well as inhibitors of the caspases suchas p35, crmA and the dominant-negative forms of the caspases. Theseforms include, for example, caspase's with an inactivating mutation ofthe active site cysteine.

[0058] Overexpression of a cell survival polypeptide can be achievedusing, for example, recombinant methods known to those skilled in theart. Routine procedures for performing such recombinant expressionmethods are described in, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1992), and in Ansubel et al., Current Protocols in Molecular Biology,John Whiley and Sons, Baltimore, Md. (1989). Such methods can be used toexpress stably or transiently a cell survival polypeptide at a levelwhich is sufficient to prevent the induction of apoptosis. The nucleicacid encoding the cell survival polypeptide can be encoded by, forexample, a homologous nucleic acid derived from the same species or celltype, or alternatively, it can be encoded by a heterologous nucleic acidderived from a different species or cell type. The source of theencoding nucleic acid is not important so long as the encoded cellsurvival polypeptide exhibits apoptosis inhibiting activity.

[0059] A level of expression of a cell survival polypeptide which issufficient to prevent the induction of apoptosis is known to thoseskilled in the art and can also be routinely determined by those skilledin the art. Expression vectors and systems are known and commerciallyavailable which provide for recombinant polypeptide expression. It is aroutine matter for one skilled in the art to choose a vector or systemwhich will provide sufficient levels of expression in a particular hostcell. Alternatively, the expression level sufficient to prevent theinduction of apoptosis can be routinely determined by expressing thecell survival polypeptide and then measuring whether the cell survivesafter treatment with an apoptotic stimulus.

[0060] In addition to recombinant methods of overexpressing a cellsurvival polypeptide, a cell can be used which inherently over expressesa cell survival polypeptide. A specific example of a cell inherentlyoverexpressing a cell survival polypeptide is the B cell lymphoma inwhich Bcl-2 was initially identified. This leukemia has a translocationof chromosome 14 to 18 causing high level expression of Bcl-2 andtherefore cell survival. The leukemic phenotype is due to the increasedcell survival. Other cell lines which inherently overexpress a cellsurvival polypeptide, either by natural or unnatural mechanisms, existand can similarly be used in the methods of the invention.

[0061] The block from apoptosis due to overexpression of a cell survivalpolypeptide and the treatment of the cells with a direct stimulus ofapoptosis provide antagonistic influences to the cell. In this way, thecells are then essentially poised for programmed cell death. A directstimulus for apoptosis can be a variety of different insults to the cellincluding, molecular, environmental and physical stimuli. As definedpreviously, such stimuli are known to those skilled in the art and canbe characterized by activating a molecule within the apoptotic pathway.Examples of direct stimuli of apoptosis include inducers such as Fasligand, anti-Fas antibody, Staurosporine, ultraviolet and gammairradiation. Thus, treatment of a cell over expressing a cell survivalpolypeptide with a direct stimulus of apoptosis will prime the cell forapoptosis since both positive and negative signals are providingbalancing effects. One advantage of this priming is that all cell deathcomponents are available for apoptosis once a single is received thatoverrides the block of the cell survival polypeptide. This advantageallows for the rapid induction of apoptosis which can be beneficial whenused to screen for compounds that possess apoptosis inducing activitywhen Bcl-2 or Bcl-xL is the cell survival polypeptide. Such cells areparticularly useful in screening for inhibitors of Bcl-2 or Bcl-xL,respectively.

[0062] In addition to treating the cells with a direct stimulus ofapoptosis, the cells are also treated with one or more compounds whichare to be tested for apoptosis inducing activity. The compounds can be,for example, small molecules, peptides, polypeptides, proteins or othermacromolecules. Essentially, the type of compound which is to be testedis unimportant, only that the user desires to test whether the compoundhas apoptotic inducing activity. Therefore, the assay is applicable fora variety of different settings, including clinical, diagnostic and drugdiscovery.

[0063] Although essentially any method which distinguishes apoptosisfrom necrosis, for example, can be used to determine cellular apoptoticactivity, the use of the single-well method described previously formeasuring apoptotic activity provides advantages. This method allows forthe rapid and efficient determination of apoptosis in what can beutilized in a multiwell or high throughput format. Therefore, once thecompounds are administered to the cell, apoptotic activity of the cellis determined by, for example, the rapid measurement of caspaseactivity, or alternatively any of a variety of other methods known inthe art. Those samples which yield positive results compared to controlsamples are indicative of compounds which will induce programmed celldeath.

[0064] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Measurement of Specific Apoptotic Activity

[0065] This Example describes the use and characteristics of thesingle-well method for the measurement of specific apoptotic activityand screening of compound libraries for inducers of apoptosis.

[0066] A human T cell leukemia clonal (Jurkat) cell line was used belowfor demonstrating and characterizing the single-well method formeasuring specific apoptotic activity of a cell. For the screening ofcompounds, this Jurkat cell line was stably transfected with anexpression construct for the cell survival polypeptide Bcl-2 and clonalcell lines were generated from the stable transfectants. The cell lineswere constructed using methods known in the art and essentially asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York (1992). Cell lines were culturedin RPMI 1640 and 10% fetal bovine serum (FBS), in the presence of amaintenance level of G418 (200 μg/ml). A neo-transfected cell line wasconstructed and cultured in a similar way for use as a control.

[0067] To ensure that the transfected cell lines were expressing Bcl-2,cell lysates were prepared from positive clones and Bcl-2 levels weremeasured by polypeptide blot analysis. Briefly, cell lysates wereprepared from 500,000 transfectants by first pelleting the culture andthen solubilizing the cells in mercaptoethanol-SDS buffer (5k (w/v)sodium dodecyl sulphate, 15% (v/v) glycerol, 0.01% (w/v) bromophenolblue, 10% (v/v) β-mercaptoethanol in 250 mM Tris-HCl, pH 6.0).Polypeptides within the lysate were separated by SDS-polyacrylamide gelelectrophoresis and transferred to nitrocellulose by electro-blotting.Detection was performed using a Bcl-2 specific monoclonal antibody thatrecognizes both endogenous and transfected forms of Bcl-2 (Hockenbery etal., Nature 348:334-336 (1990)). Expression levels were quantified byusing different amounts of purified recombinant Bcl-2 run on the samegel as a comparison. Comparison of the transfected Bcl-2 expressionlevels with the known amounts indicated an expression level of about 6ng/10⁶ cells. The expression level of endogenous Bax, detected with ananti-Bax specific antibody and quantified in a similar manner wasdetermined to be about 1 ng/10⁶ cells (Reed et al., Analytical Biochem.205:70-76 (1992)).

[0068] To induce apoptosis, either a neo-transfected Jurkat cell line orthe above Bcl-2 transfected cell line was used. Specifically, the celllines were harvested, washed in RPMI 1640 (without phenol red as anindicator) and 10% FBS, and resuspended at a density of 1.33×10⁶cells/ml in the same medium. Cells were plated in 96-well plates at100,000 cells/well in a 75 μl volume and allowed to acclimatise for 1hour at 37° C. Apoptosis was induced by incubation with an anti-Fasantibody which was added to the cells to a final concentration of 100ng/ml and allowed to incubate for 3 hours at 37° C. (11 μl of a 10×stocksolution; MBL, Pavera: Madison, Wis.; Yonehara et al., Experimental Med.169:1747-1756 (1989)). Alternatively, for screening procedures,compounds were pre-incubated with the Bcl-2 transfected cells for 1 hourat 37° C. prior to the addition of anti-Fas antibody. Compounds to betested were added to duplicate wells at a 4×concentration (25 μl)resulting in a final concentration of 5 μg/ml compound and 1% DMSO.

[0069] Apoptosis was determined by measuring aspartic specific cysteineprotease (caspase) activity following anti-Fas antibody incubation.Briefly, the cells were lysed by the addition of 10×lysis buffer andshaking in an orbital shaker for 5 min at room temperature (12.3μl/well; 2 mM PMSF, 10 mM DTT, 10 μg/ml Pepstatin A, 10 μg/ml Leupeptin,50 μg/ml Aprotenin, 1 mM EDTA, 1 mM EGTA and 5% CHAPS in 10×hypotonicbuffer (100 mM Hepes pH 7.4, 420 mM KCl, 50 mM MgCl₂)). ICE buffer wassubsequently added (70 μl/well of a 2×stock prepared as follows: 40 mMHepes, pH 7.5, 2 mM EDTA, 20% Sucrose and 0.2% CHAPS to which DTT wasadded fresh before the start of every assay to a final concentration of10 mM) and the enzyme assay was initiated by addition of the DEVD-AMCsubstrate to a final 2 μM concentration (10 μl of a 20×stock).Alternatively, the lysis buffer, ICE buffer and DEVD-AMC substrate canbe combined into a single buffer and added in one step. Substratecleavage activity was measured at thirty minute intervals over a periodof two hours beginning at time zero in a fluorescent plate reader. Theexcitation wavelength was 360/40 nm and the emission wavelength 460/40nm.

[0070] Controls run on each multiwell plate included a matchingneo-transfected cell line treated with anti-Fas antibody, but nocompound, as the positive death control and Bcl-2 transfected cells withand without antibody as the background controls.

[0071] The characterization and use of the above method for screeninginducers of apoptosis is described further below.

[0072] Studies were initially carried out comparing the single-wellmethod described above (where the entire experiment is carried out inthe same well), with a known method where cells are spun down and washedafter anti-Fas antibody treatment and before addition of lysis buffer.This comparison is presented in FIG. 1A. The results show that bothmethods yield comparable caspase activity as demonstrated by theCPP32-like enzymatic cleavage of the DEVD-AMC substrate inneo-transformed cell lines. In contrast, cell lines transfected withBcl-2 are protected from Fas induced death and show little increase incleavage (FIG. 1B). These results demonstrate that the single-wellmethod can accurately measure both the induction and inhibition ofcaspase activity at a sensitivity comparable to previously used methods.

[0073] Further characterization of the single-well method demonstratedthat the presence of 10% FBS was beneficial throughout the course of theassay, up to lysis of the cells. For example, substitution with 0.5%bovine serum albumin resulted in induction of CPP32-like activity in theneo-transfected Jurkat cells in the absence of anti-Fas antibodytreatment. This effect is likely due to induction of apoptosis throughgrowth factor withdrawal. However, the expression of Bcl-2 was able toprotect the cells from this effect.

[0074] Further, compounds in libraries for high throughput screening areroutinely maintained as stock solutions dissolved in DMSO. Furthercharacterization of the single-well method was carried out to determinepermissible levels of DMSO permissible in the method. The resultsindicated that up to at least 1% DMSO had no significant effect on thecells or the accuracy of the method. Testing of the possible effect ofthe media component phenol red, which is normally used as an indicatorin RPMI, had a slight, but significant, quenching effect on thefluorescence readings. Due to this effect, phenol red was removed fromthe media in all further assays.

[0075] To optimize the incubation time and concentration of anti-Fasantibody, studies were performed which measured the caspase activityover a range of incubation time points and concentration of anti-Fasantibody. Caspase activity was determined in both neo- and Bcl-2transfected cells. In the neo-transfected cells, induction of aCPP32-like activity increased with anti-Fas antibody concentration up to30 ng/ml, after which a plateau was reached. A slight, but significant,increase in CPP32-like activity was observed in the Bcl-2-transfectedJurkats at an antibody concentration of 100 ng/ml.

[0076] At anti-Fas antibody concentrations above 10 ng/ml the highestenzymatic activity was observed between 2 and 5 hours after addition ofthe antibody to neo-transfected Jurkat cells. Longer incubation timesresulted in a decrease of observable CPP32-like activity (FIG. 2A). InBcl-2 transfected cells, what little enzymatic activity was detected wasseen only at an incubation time of 4 hours (FIG. 2B). From the aboveresults an antibody concentration of 100 ng/ml incubated for 3 hours,was chosen as the standard running conditions for the assay.

[0077] Further characterization of the assay was performed by assessingboth the intra-assay and inter-assay variation. Specifically, theintra-assay variation of the procedure was examined by running 12 wellsof each of the neo-transfected and of the Bcl-2 transfected cell lineson the same plate at the same time in the presence or absence ofanti-Fas antibody. The results of this study are shown in Table 1 andreveal an overall intra-assay variation of 6.8% or less of the mean ofthe caspase activity of the positive, neo-transfected control treatedwith anti-Fas. TABLE 1 Intra-assay variation (RFU) Mean Std Dev % CVNeo-transfected + anti-Fas antibody 228.3 15.6 6.8 Neo-transfected −anti-Fas antibody 17.7 4.3 1.9 Bcl2-transfected + anti-Fas antibody 10.23.3 1.4 Bcl2-transfected − anti-Fas antibody 2.4 3.5 1.5

[0078] Alternatively, the inter-assay variation was determined byrunning each of the neo- or Bcl-2 transfected cell lines 24 on separateplates over the course of two days. Each plate was set up individually.The result of this determination are presented in Table 2 and reveal anoverall inter-assay variation of 7.0% or less of the mean of the caspaseactivity of the positive, neo-transfected control treated with anti-Fas.TABLE 2 Inter-assay variation (RFU) Mean Std Dev % CV Neo-transfected +anti-Fas antibody 221.5 15.5 7.0 Neo-transfected − anti-Fas antibody 7.72.9 1.3 Bcl2-transfected + anti-Fas antibody 22.4 8.2 3.7Bcl2-transfected − anti-Fas antibody −1.5 2.0 0.9

EXAMPLE II Screening Compounds for the Induction of Apoptosis

[0079] This Example describes the use of the screening of a compoundlibrary for the detection of apoptotic inducers.

[0080] The single-well method for determining specific apoptoticactivity was used in conjunction with Bcl-2 transfected cells toidentify compounds that exhibit apoptotic inducing activity. Theprocedures and the Bcl-2 transfected cell line was described above inExample I.

[0081] Briefly, 960 compounds chosen from a chemical library wereexamined for their ability to induce apoptosis in cells that were primedby the incubation of Bcl-2 stably transfected cells with anti-Fasantibody. The screening was performed in a multiwell or high throughputmode and the compounds were incubated at a concentration of 5 μg/ml. Theresults of this screen are shown in FIG. 3. Values are expressed as apercentage of caspase activity of the positive, neo-transfected controltreated with anti-Fas present on each plate. The levels of caspaseactivity ranged from 62.1 to −11.5% of the non-Bcl-2 protected control.A mean value of 1.7% and a standard deviation of 4.2% was also observed.

[0082] The threshold for defining a compound as positive was set at 24%of the caspase activity of neo-transfectants in the absent of anycompound (46 relative fluorescence units). This number was determined bythe mean plus 3 standard deviations of the negative controls (anti-Fastreated Bcl-2-transfected cells) shown in Table 2 for calculating theinter-assay variation. This threshold is valid assuming a normaldistribution for the results. Given this assumption, the probabilitythat a negative compound will exceed the mean of the negative controlsby more than 3 standard deviations is 0.0028. Using this criterion, 5positives were identified from the 960 compounds screened.

[0083] Throughout this application various publications have beenreferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order todescribe more fully the state of the art to which this inventionpertains.

[0084] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A single-well, microscale method of determiningthe specific apoptotic activity of a cell comprising contacting a cellpopulation of about 1×10⁵ cells for a time period of between about 30minutes and 4 hours with a sufficient volume of medium containing anapoptotic specific diagnostic reagent and a diagnostic accessory reagentso as to cover said cell population, and determining the activity ofsaid apoptotic specific diagnostic reagent.
 2. The method of claim 1,wherein said cell population further comprises greater than about 10,000cells, more preferably about 50,000 cells, and preferably about 100,000cells.
 3. The method of claim 1, wherein said time period furthercomprises between about 30 minutes to 4 hours, preferably between about30 minutes to 2 hours, and more preferably about 1 hour.
 4. The methodof claim 1, wherein said volume further comprises between about 1 and200 μl, preferably between about 30 and 125 μl, more preferably about100 μl.
 5. The method of claim 1, wherein said apoptotic specificdiagnostic reagent further comprises an caspase specific substrate orAnnexin V.
 6. The method of claim 5, wherein said diagnostic accessoryreagent is a lysis reagent or calcium.
 7. The method of claim 1, whereinsaid apoptotic specific diagnostic reagent further comprises an caspasespecific substrate attached to a detectable label.
 8. The method ofclaim 1, wherein said apoptotic specific diagnostic reagent is selectedfrom the group consisting of ZEVD-AMC, YVAD-AMC and DEVD-AMC.
 9. Themethod of claim 1, further comprising a multiwell format.
 10. The methodof claim 1, further comprising a plurality of different single-wells forthe simultaneous determination of multiple different samples.
 11. Amethod of identifying a compound which induces apoptosis comprising: (a)providing a cell overexpressing a cell survival polypeptide, said cellsurvival polypeptide being overexpressed at a level which is sufficientto prevent the induction of apoptosis; (b) treating said celloverexpressing said cell survival polypeptide with a direct stimulus ofthe cell death pathway; (c) adding a compound to be tested for apoptoticinducing activity, and (d) determining cellular apoptotic activity,wherein the presence of apoptotic activity is indicative of saidcompound being an apoptotic inducer.
 12. The method of claim 11, whereinsaid cell survival polypeptide comprises Bcl-2, Bcl-xL, Mcl-1 andE1B-19K.
 13. The method of claim 11, wherein said cell survivalpolypeptide is overexpressed at a level so as to prevent the inductionof a direct stimulus of the cell death pathway.
 14. The method of claim13, wherein said cell survival polypeptide is overexpressed at a levelso as to prevent the induction of apoptosis by a direct stimulusselected from the group consisting of Fas ligand, anti-Fas antibody,staurosporine, UV and gamma irradiation.
 15. The method of claim 11,wherein said direct stimulus of the cell death pathway is selected fromthe group consisting of Fas ligand, anti-Fas antibody, staurosporine, UVand gamma irradiation.
 16. The method of claim 11, wherein said cellsurvival polypeptide is encoded by an exogenous nucleic acid.
 17. Themethod of claim 16, wherein said exogenous nucleic acid is either ahomologous or heterologous nucleic acid.
 18. The method of claim 11,wherein said cell survival polypeptide is encoded by an endogenousnucleic acid.
 19. The method of claim 11, wherein said compound testedfor apoptotic inducing activity further comprises a compound whichinduces caspase activity in a cell.
 20. The method of claim 11, whereinsaid compound tested for apoptotic inducing activity further comprises acompound which inhibits the activity of a cell survival polypeptide. 21.The method of claim 11, wherein said compound tested for apoptoticinducing activity further comprises a compound which promotes theactivity of a cell death polypeptide.
 22. The method of claim 11,wherein said apoptotic activity in step (d) further comprises lysingsaid cells and determining the caspase activity in said lysate.
 23. Themethod of claim 11, wherein said apoptotic activity in step (d) furthercomprises contacting the cells with Annexin 5 and determining the amountof Annexin 5 which binds.
 24. A rapid method of identifying a compoundwhich inhibits apoptosis comprising: (a) separately contacting aplurality of cell populations with a different compound to be tested forapoptotic inhibiting activity; (b) incubating said cells with a directstimulus of the cell death pathway for a period of between about 2minutes to 3 hours, and (c) measuring the specific apoptotic activity ofthe cells.
 25. The method of claim 24, wherein said direct stimulus ofthe cell death pathway is selected from the group consisting of Fasligand, anti-Fas antibody and staurosporine UV and gamma irradiation.26. The method of claim 24, wherein step (c) further comprises lysingsaid cells and determining caspase activity in said lysate.
 27. Themethod of claim 24, wherein said compound exhibits caspase inhibitoryactivity.
 28. The method of claim 24, wherein said compound promotes theactivity of a cell survival polypeptide.
 29. The method of claim 24,wherein said compound exhibits cell death polypeptide inhibitoryactivity.